The present invention relates to a novel method for the diagnosis of Lyme borreliosis, or more specifically a method for detecting antibodies directed against the OspC protein of Borrelia burgdorferi sensu lato. Further, the invention pertains to an immunological agent which comprises a specific peptide fragment derived from the C-terminus of OspC and uses of this immunological agent in the diagnosis of Lyme borreliosis as well as for vaccination purposes. The invention finally relates to novel polypeptide fragments derived from the C-terminus of OspC as well as to short peptides derived from this region.
The tickborne spirochaete Borrelia burgdorferi is the etiological agent of Lyme borreliosis, which is at present the most common vector-borne human disease in Europe and North America. Lyme borreliosis is a common tick-borne disease which is caused by one of the three genospecies of B. burgdorferi sensu lato: B. burgdorferi sensu stricto, B. garinii, and B. afzelii. The clinical manifestations are diverse and may involve the skin, central nervous system, heart, and joints. The symptomatology can be divided into three stages: The first stage: skin lesion; the second stage: meningitis, arthritis, and myocarditis; the third stage: chronic meningitis, chronic arthritis, and chronic skin lesion.
It is desirable to have access to an assay with a high diagnostic sensitivity already in the first stage of Lyme borreliosis, in order to diagnose and treat patients before they develop severe symptoms of the later stages of Lyme borreliosis.
Laboratory diagnosis of Lyme borreliosis has been possible since the discovery of B. burgdorferi in 1982. However, the ultimate diagnostic assay has not yet been developed. Laboratory confirmation of Lyme borreliosis still relies mainly on the detection of antibodies to B. burgdorferi. Assays based on whole cell B. burgdorferi extracts lack diagnostic specificity due to antibodies cross-reacting with antigens from a wide range of bacterial species. Western blotting (WB) has proved difficult to perform due to strain differences, the complexity of the band patterns, and inherent problems in standardization of Western blotting in general. Efforts have therefore mainly been directed towards identification of single immunodominant antigens, either in the native form or as recombinant proteins, which can be purified and used as test antigens.
According to Western blot studies there are only two B. burgdorferi antigens that meet the essential criterium of eliciting an early and strong antibody response in the majority of patients. These are the B. burgdorferi flagellum and the outer surface protein C (OspC). Whereas the performance of EIA""s using purified native B. burgdorferi flagellum is well documented, the reported experience with OspC EIA""s is still limited.
Other routes to the specific diagnosis of Lyme borreliosis have been suggested. A fraction of membrane related proteins and lipids known as xe2x80x9cfraction Bxe2x80x9d disclosed in EP-A-445,135 has been demonstrated to exhibit an improved diagnostic specificity, but the provision of fraction B requires that Borrelia burgdorferi sensu lato is cultured and subsequently treated in a series of steps.
A high prevalence of IgM anti-OspC antibodies has been found in patients in the two first stages of Lyme borreliosis by means of Western blotting, using native and recombinant OspC (rOspC) and by means of ELISA, using rOspC (Fung B. P. et al. (1994); Gerber M. A. et al. (1995); Wilske B. et al. (1994); Padula S. J. et al. (1994)).
In general, it has been concluded by the present inventors that the sensitivity of diagnosis of the early stages of Lyme borreliosis could be increased by combining the results from an immunoassay based on the detection of anti OspC antibodies and the results from the current available immunoassays for the flagellum.
More specifically, the present inventors have reached the conclusions that certain C-terminal fragments of OspC comprise an epitope which is essential in the human immune system""s recognition of OspC. Additionally, it has been found that the serodiagnostic sensitivity of said C-terminal fragments is surprisingly high when compared to that of full-length OspC.
These conclusions have been reached after immunological experiments which surprisingly have revealed that 1) a synthetic peptide derived from the C-terminus of OspC of B. burgdorferi sensu lato exhibits an immunological sensitivity in detecting sera from human Lyme borreliosis patients which is at least 85% of the sensitivity of full length recombinant B. burgdorferi sensu lato derived OspC (rOspCfl) when used in similar assays, and 2) a recombinant B. burgdorferi sensu lato OspC truncate which lacks the 7 carboxyterminal amino acids (rOspCt) exhibits, when compared to full length recombinant OspC (rOspCfl), a very poor, if any, immunological reactivity with sera from patients suffering from Lyme borreliosis.
These immunological experiments were part of scientific work which aimed at producing an immunoassay based on recognition by antisera of recombinant OspC. However, in the first attempt, a diagnostic sensitivity of less than 5% was achieved in early stage of Lyme borreliosis (this involves the first and second stage of Lyme borreliosis), cf. Example 1. Here, the deduced amino acid sequence of three OspC proteins representing each of three B. burgdorferi genospecies (B. burgdorferi sensu stricto, B. garinii, and B. afzelii) were used as test antigens. However, the recombinant proteins all lacked the seven C-terminal amino acid residues, because these had not yet been determined for the three pertinent isolates of Borrelia burgdorferi sensu lato.
In the second attempt the entire recombinant OspC proteins (rOspcfl) from all three strains were produced, including the last seven amino acid residues, which had then been deduced. Further, the deduced amino acid sequence in the C-terminus of the OspC protein was identical for the genospecies of B. garinii and B. afzelii used in the first attempt, whereas the B. burgdorferi sensu stricto genospecies had a valine residue instead of an alanine residue in position 205. By employing the rOspCfl proteins as test antigens in an ELISA, diagnostic sensitivities were achieved of 44% for IgM in the first stage of Lyme borreliosis and 48% for IgM in the second stage of Lyme borreliosis in a set of preliminary tests. The diagnostic sensitivity for borreliosis was identical for all three genospecies. Therefore, a more comprehensive testing of the immunological reactivity of rOspCfl and of synthetic C-terminus derived peptides were performed, cf. Example 2.
On the background of these findings, it was concluded that the seven carboxy-terminal amino acid residues comprise, constitute, or form part of an antigenic epitope which is essential in the human immunological recognition of OspC and it was therefore conceived that this epitopic region can be the basis for novel and improved diagnostic means.
It was further investigated to what degree each of the single amino acids contributes to the immune reactivity of the C-terminus of OspC, and it was found that the last 5 amino acids can only be varied to a very limited degree, whereas e.g. alanine substitutions in other amino acids in the C-terminus had no or little impact on immune reactivity.
A number of advantages can be provided by using short OspC fragments as part of an immunological agent in the diagnosis of early stage Lyme borreliosis. Most important, an immunoassay, such as an ELISA, which is based on a synthetic peptidexe2x80x94as opposed to using full-length or near-full-length OspCxe2x80x94simplifies the preparation and purification steps of the components of the assay and thus helps standardize the assay.
Further, the use of a short peptide in an immunoassay may lead to a decrease in the cross-reactivity with antibodies raised against other antigens as a consequence of the abolishment of a large number of potentially cross-reacting epitopes in OspC (for instance, the present peptides lack sequence homology with the variable membrane proteins of B. Hermsii). On the other hand, the use of an antigen, such as full-length OspC, which comprises a significant number of epitopes normally has as a result that the signal from a cross-reacting epitope may be xe2x80x9cdrownedxe2x80x9d in the signals from other epitopes, an effect which cannot be expected from an antigen comprising only a few epitopes. Therefore, the short peptide should preferably exhibit a very specific pattern of immunological reaction with antibodies against other antigens, cf. the discussion of specificity below.
As test antigen in an immunoassay, the peptide of the invention may prove to exhibit a superior diagnostic sensitivity in the early stage of Lyme borreliosis compared to e.g. an rOspCfl ELISA. This is due to the fact that the relatively small size of the peptide of the invention allows binding of a large number of peptides to the solid surface of the ELISA without the side effect that these peptides interfere with each other, whereas the relatively large rOspCfl molecules may indeed interfere with themselves and each other and e.g. mask epitopes which could potentially react with antibodies.
Finally, even though it would be expected that the use of a short peptide would lead to a marked decrease in sensitivity when testing patient antisera (which by nature are polyclonal), the present inventors have demonstrated that short peptides exhibit a high sensitivity when compared to the rOspCfl (cf. Example 2).
Therefore, peptide fragments derived from the C-terminus of Borrelia burgdorferi sensu lato OspC will according to the invention serve as diagnostic tools in the diagnosis of Lyme borreliosis.
In its broadest aspect, the invention therefore relates to a method for determining previous or ongoing sensitization of a subject with OspC polypeptide of Borrelia burgdorferi sensu lato, said method comprising contacting immunoglobulins or T-cells derived from the subject with at least one immunological agent comprising a polypeptide fragment which contains a peptide having a degree of sequence identity of at least 50% with a Borrelia burgdorferi sensu lato derived peptide which either has the amino acid sequence SEQ ID NO: 1:
Pro-Val-Val-Ala-Glu-Ser-Pro-Lys-Lys-Pro
or has a subsequence of SEQ ID NO: 1 which has a length of at least 5 amino acid residues, and subsequently detecting the degree, if any, of immunological reactivity between the immunoglobulins and the immunological agent or between the T-cells and the immunological agent, a significant immunological reaction being indicative of previous sensitization with OspC polypeptide from Borrelia burgdorferi sensu lato, in which method said polypeptide fragment
a) is one which, when used in a first ELISA (the xe2x80x9cpeptide ELISAxe2x80x9d described in the Example section), results in an immunological average sensitivity in detecting randomly selected antisera from patients suffering from early stage Lyme borreliosis which is at least 75% of the average immunological sensitivity in detecting the same antisera in a second ELISA (the xe2x80x9crOspC ELISAxe2x80x9d described in the Example section) using full-length recombinant OspC derived from Borrelia garinii, and/or
b) has a length of at most 60 amino acid residues.
(In short, the first ELISA can be performed as follows:
i) coating flat-bottom microdilution plates with 100 xcexcl of streptavidin (2.5 xcexcg/ml) in citrate buffer (pH 5) and incubating overnight at 4xc2x0 C.,
ii) washing the plates four times for one minute with phosphate buffered saline (PBS) containing 0.5 M NaCl and 0.1% (vol/vol) Tween 20 (pH 7.2),
iii) adding to each well 100 xcexcl of biotinylated polypeptide fragment which is prodiluted in PBS containing 0.37 M NaCl, 0.5% (vol/vol) Tween 20, and 1% (wt/vol) milkpowder (pH 7.0)) and incubating the plates overnight at 4xc2x0 C.,
iv) washing the plates four times for one minute with PBS containing 0.5 M NaCl and 0.1% (vol/vol) Tween 20 (pH 7.2),
v) adding 100 xcexcl of serum diluted 1:200 in PBS containing 0.7 M NaCl, 0.1% (vol/vol) Tween 20, and 1% (wt/vol) milkpowder (pH 7, 2) to each well and incubating for 2 hours at 20xc2x0 C. on a rocker platform,
vi) washing the plates four times for one minute with PBS containing 0.5 M NaCl and 0.1% (vol/vol) Tween 20 (pH 7.2),
vii) adding 100 xcexcl of peroxidase conjugated rabbit anti-human IgM diluted 1:1000 in PBS containing 0.5% Tween 20 and 1% milkpowder (pH 7.4) to each well and incubating for 1 h at 20xc2x0 C.,
viii) washing the plates four times for one minute with PBS containing 0.5 M NaCl and 0.1% (vol/vol) Tween 20 (pH 7.2),
ix) adding 200 xcexcl of o-phenylenediamine (0.33 mg/ml) dissolved in citrate buffer (pH 5) with 0.04% (vol/vol) H2O2 to each well and protecting the plates from light for 15 minutes,
x) stopping the enzymatic reaction by adding 50 xcexcl of 3 M H2SO4 to each well,
xi) reading the optical density (OD) at 492 nm for each well,
xii) if two OD values the same serum sample differs more than 10% from the mean, retesting said sera samples by steps i-xi, and
xiii) establishing as a result that an OD of at least 0.460 is a significant immunological reaction whereas an OD of less than 0.460 is a negative reaction;
similarly, the second ELISA can be performed as follows:
I) coating flat-bottom microdilution plates with 100 xcexcl of an optimum coating concentration of full length rOspC (rOspCfl), diluted in 0.05 M bicarbonate pH 9.6, for 1 hour at 20xc2x0 C. on a rocker platform and thereafter overnight at 4xc2x0 C.
II) washing the plates four times for one minute with phosphate buffered saline (PBS) containing 0.5 M NaCl and 0.1% (vol/vol) Tween 20 (pH 7.2),
III) blocking unspecific protein binding with 100 xcexcl 3% (wt/vol) milk powder in PBS for 1 hour,
IV) washing the plates four times for one minute with PBS containing 0.5 M NaCl and 0.1% (vol/vol) Tween 20 (pH 7.2),
V) adding 100 xcexcl of serum diluted 1:200 in PBS containing 0.1% (vol/vol) Tween 20, 0.02% NaN3 and 1% (wt/vol) milk powder to each well and incubating for 2 hours at 20xc2x0 C.,
VI) washing the plates four times for one minute with PBS containing 0.5 M NaCl and 0.1% (vol/vol) Tween 20 (pH 7.2),
VII) adding 100 xcexcl of peroxidase conjugated rabbit anti-human IgM diluted 1:1000 in PBS containing 0.5% Tween 20 and 1% milkpowder (pH 7.4) to each well and incubating for 1 h at 20xc2x0 C.,
VIII) washing the plates four times for one minute with PBS containing 0.5 M NaCl and 0.1% (vol/vol) Tween 20 (pH 7.2),
IX) adding 200 xcexcl of o-phenylenediamine (0.41 mg/ml) dissolved in citrate buffer (pH 5) with 0.04% (vol/vol) H2O2 to each well and protecting the plates from light for 15 minutes,
X) stopping the enzymatic reaction by adding 50 xcexcl of 3 M H2SO4 to each well,
XI) reading the optical density (OD) at 492 nm for each well,
XII) if two OD values the same serum sample differs more than 10% from the mean, retesting said sera samples by steps i-xi, and
XII) establishing as a result that an OD of at least 0.230 is a significant immunological reaction whereas an OD of less than 0.230 is a negative reaction.)
In conclusion, the present invention thus provides short (normally synthetic) peptides which bind anti-Borrelia antibodies in serum from patients with early stage Lyme borreliosis. These peptides can be used together with different means enabling the easy detection of Borrelia burgdorferi sensu lato infection. A serodiagnostic assay based on the use of these peptides, optionally combined with other antigens of B. burgdorferi increases the total diagnostic sensitivity. Accordingly the patients can be treated before they develop symptoms in the central nervous system.